5-methylcytosine (5-mC) has been linked to gene expression and its distribution in the genome plays an important role in epigenetics. In 2009, two groups independently discovered that an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC), exists in human and mouse DNA, and is especially enriched in the neuronal tissues as well as embryonic stem cells. Three enzymes named TET1/2/3 have been shown in human and mouse to be responsible for oxidizing 5-mC to 5-hmC. TET enzymes belong to the broad family of Fe(II)/2-oxo-glutarate-dependent (2OGFE) oxygenases, which use 2-oxo-glutarate (2OG), as co-substrate, and ferrous ion (Fe(II)) as cofactor. After additional biochemical studies, it was discovered that these enzymes could oxidize 5-mC to generate oxidation products identified as 5-hmC, 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC). Finally, 5-caC is believed to be excised via the action of DNA glycosylases and replaced by the unmodified cytosine. The TET enzymes are very large proteins and hence it has been problematic to make these proteins in recombinant form and in sufficient quantities to use as a research reagent.
In order to identify the impact of the epigenome on phenotype, it is desirable to map the position of modified nucleotides and to understand when and where the various modifications arise. Sodium bisulfite sequencing is the predominant method for mapping modified cytosine in the genome. Unfortunately, this technique does not discriminate between 5-mC and 5-hmC. Different methods are required to distinguish 5-mC from 5-hmC and its oxidation products.